JXB Advance Access originally published online on February 27, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 919-927, April 1, 2004
© 2004 Oxford University Press
Plants and the Environment |
Effects of a PAL inhibitor on phenolic accumulation and UV-B tolerance in Spirodela intermedia (Koch.)
Received 3 September 2003; Accepted 19 December 2003
1 Cropping Systems Research Laboratory, ARS-USDA, Lubbock, TX 79415, USA
2 Natural Resource Sciences and Landscape Architecture, University of Maryland, College Park, MD 20741, USA
3 Department of Botany; 392 Pearson Hall; Miami University, Oxford, OH 45056, USA
* To whom correspondence should be addressed. Fax: +1 513 529 4243. E-mail: huertaaj{at}muohio.edu
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Abstract |
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Duckweed (Spirodela intermedia) was grown axenically on 1/2 strength Hutner’s nutrient solution plus 1% sucrose, with the L-phenylalanine ammonia-lyase (PAL) inhibitor 2-aminoindan-2-phosphonic acid (AIP) at 0.0, 0.05, or 10 µM, at constant 25 °C and a light intensity of 300 µmol m–2 s–1 photosynthetically active radiation from CW fluorescent lamps. Growth with 10 µM AIP led to decreased frond area and fresh weight, but dry weight was unchanged. Microscopic examination of fronds revealed increased frond thickness and a lack of reticulate aerenchyma. Ultraviolet epifluorescence microscopy and UV-Vis spectroscopy of methanolic extracts confirmed the dose-dependent inhibition of secondary phenolic synthesis with the near total elimination of secondary phenolic accumulation at the 10 µM level. AIP-treated plants showed increased sensitivity to UV-B as shown by a reduced Fv/Fm. The results provided direct evidence of the working hypothesis that phenols function to screen UV radiation from reaching photosynthetic tissues or damaging other sensitive tissues. A novel histochemical method employing zirconyl chloride to visualize phenols is discussed.
Key words: AIP, aminophosphonic acid, duckweed, flavonoid, PAL, phenolic, Spirodela, ultraviolet-B, UV.
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Introduction |
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Secondary phenolic compounds, notably flavonoids and hydroxycinnamic acids, exhibit high UV absorptivities, are expressed to a great extent in the epidermis, and are virtually ubiquitous in tracheophytes (McClure, 1975). This has led to the long-standing hypothesis that a primary adaptive advantage conferred by these compounds is that they absorb potentially harmful UV-B radiation (280–315 nm) at the leaf surface and protect underlying photosynthetic tissues. Direct evidence of the role of phenolic accumulation in conferring UV tolerance has been obtained in Arabidopsis thaliana (L.) Heynh. mutants deficient in specific secondary phenolic biosynthetic enzyme activities. This allowed determination of the relative importance of phenylpropanoids and flavonoids in conferring UV tolerance in A. thaliana (Sheahan, 1996). Direct evidence using similar methods has been obtained in only a very few other plants.
Most evidence for the role of phenolics as UV screens has been correlative. Generally, when plants are allowed to develop under enhanced UV-B regimes or allowed to acclimate to doses of supplemental UV-B radiation, soluble phenolics and photosynthetic tolerance to UV increase simultaneously. In addition to increased phenolics, other presumably adaptive responses have been observed. These include increases in leaf thickness, free radical scavenging capacity, PSII protein turnover rates, and altered chlorophyll and carotenoid content (Jansen, 2002; Jansen et al., 1998).
In duckweeds, it has been difficult to factor out the relative importance of secondary phenolics as UV screens, because of constitutive tolerance to UV-B, the presence of alternative protective mechanisms, and a lack of mutants devoid of, or at least deficient in, aspects of secondary phenolic metabolism. The inherent UV-B tolerance of duckweed was shown in experiments where Lemna minor L. was grown under low level (150 µmol m–2 s–1) of PAR (photosynthetically active radiation, 400–700 nm), and irradiated with high levels of UV-B. Although growth under such low light levels typically increases plant sensitivity to UV-B, little effect on PSII was noted (Germ and Gaber
ik, 1999). In Spirodela punctata clones, variation in extractable flavonol content was not related to UV tolerance (Jansen et al., 1999). Peroxidase activity, auxin metabolism, increased free radical scavenging, and increased lignification have all been implicated in UV-B stress protection in Spirodela punctata and Lemna gibba (Jansen et al., 1996a, b, 2001).
The primary goal of this work was to examine the role of secondary phenols in conferring UV-B tolerance in Spirodela intermedia without resorting to the more conventional process of inducing, selecting, characterizing, and describing mutants. By growing the plants in the presence of 2-aminoindan-2-phosphonic acid (AIP) it was possible to produce plants with highly reduced secondary phenolic metabolism throughout their development. AIP is a highly-specific inhibitor of the enzyme L-phenylalanine-ammonia-lyase (PAL) which initiates all secondary phenolic biosynthesis (Zon and Amrhein, 1992). More over, duckweed was selected because it was thought that it would be possible to produce plants devoid of phenylalanine ammonia-lyase activity while simultaneously avoiding problems associated with the inhibition of lignin and suberin synthesis. The duckweeds grow in a prostrate orientation upon the surface of water and rely on the buoyant forces of their environment for support, rather than lignified structures. Duckweeds also have the advantage that they may be grown in axenic cultures, reproducing vegetatively. Duckweeds have been extensively characterized insofar as their photosynthetic apparatus is concerned and particularly with regard to the response of PSII to UV-B (Jansen et al., 1996a). Spirodela intermedia was chosen in particular because it is the largest known duckweed (McClure and Alston, 1966). Also, the flavonoid chemistry (McClure and Alston, 1966), photocontrol of flavonoids (McClure, 1968) and lignification characteristics (Blazey and McClure, 1968) of this clone have been described.
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Materials and methods |
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Synthesis of 2-aminoindan-2-phosphonic acid (AIP)
AIP was synthesized essentially as outlined in Zon and Amrhein (1992). Briefly, the reaction proceeds by the addition of triethylphosphonyl acetate to
,’ dibromo-o-xylene driven by successive sodium ethanolate deprotonation of the carbon ß to the carbonyl carbon and phosphonyl phosphorus of the triethylphosphonyl acetate. The resulting ester was saponified under basic conditions to the free acid, converted to the acyl chloride with thionyl chloride, and then to the amide with anhydrous ammonia gas. A Hoffman rearrangement employing sodium hypobromite yielded the amine which was acid hydrolysed to obtain crude 2-aminoindan-2-phosphonic acid (AIP). After successive recrystallizations, the identity of the final purified product was confirmed as AIP by comparison of IR spectroscopy and melting point determination of the synthesized compound against the published values (Zon and Amrhein, 1992) and against an authentic 10 mg sample generously provided by Professor Amrhein (Institute of Plant Sciences, ETH-Zurich Universitätstrasse 2, CH-8092 Zürich, Switzerland). Biological activity was confirmed by its ability to inhibit anthocyanin synthesis in red cabbage (Brassica oleracea var. capitata L.) seedlings (Gitz III et al., 1998).
Plant material
The Spirodela intermedia clone used in this work was originally collected in Uruguay and its flavonoid chemistry described in McClure and Alston (1966) where it was designated as clone 115. This clone was then maintained by Elias Landolt, ETH, Zurich, Switzerland, who generously provided the plant for this work. Axenic cultures were grown in 2.0 l low-form culture flasks on 500 ml of half-strength Hutner’s nutrient solution with 1% sucrose (McClure, 1968) plus 0.0, 0.5, or 10 µM l–1 AIP. The nutrient solution pH was adjusted to 6.0 prior to autoclaving. Cultures were initiated by the introduction of two fronds and allowing the cultures to develop in chambers at constant 25 °C and a light intensity of 300 µmol m–2 s–1 provided by cool white fluorescent lamps. Plants were harvested after 6 weeks. At this stage the surface of the medium within the flask was generally covered with plants.
Scanning electron microscopy
Fronds were fixed for 4 h in 0.1 M phosphate buffer (pH 7.2) plus 4% glutaraldehyde, taken through an alcoholic dehydration series, fractured under liquid nitrogen, critical point dried under carbon dioxide, mounted on aluminium stubs, and sputter-coated with gold to a thickness of about 25 nm (Bornman and Chen, 1993). The specimens were observed in a JEOL Model T-200 at an accelerating voltage of 15 kV and the images photographed onto Polaroid 55 P/N film.
Optical microscopy
Freshly harvested plants were sectioned with a vibrating microtome (VibratomeTM Series 1000, Technical Products International, Inc., St Louis, MO). The resultant 100 µM thick sections were viewed on a Nikon Microphot microscope at 10x with and without UV fluorescence excitation from a Hg-Xe arc lamp and images were photographed using Ektachrome 200 ASA film. Because the authors were interested in localization and qualitative differences in phenolic expression, rather than quantification by fluorescent intensities, exposure times were not held constant between treatments. Phenolics were visualized by UV epifluorescence optical microscopy using the ‘natural product reagent’ (NP, 2-aminoethyl diphenylborinate) (Hutzler et al., 1998), 1% (w/v) aqueous zirconium chloride (Zr) solution (Welcher, 1948; Seikel, 1962) and fuming with NH3 (Hartley and Harris, 1980). Since the NP reagent is practically insoluble in water a 1% ethanolic solution was used. NP and Zr were applied to water-mounted sections by drawing the histochemical solutions under and across the cover slips. NH3 fuming was done by holding water mounts over paper toweling saturated with strong aqueous ammonium hydroxide solution in covered Petri dishes.
Photosynthetic and phenolic pigment determination
Duckweed colonies were placed in disposable glass tubes, covered with 10 ml of extraction solvent (MeOH:H2O:AcOOH, 50:50:1 by vol.), carefully sealed with several layers of laboratory film, placed in the dark at room temperature (20–23 °C) and agitated gently once each day for 3–5 d. Use of 50% methanol allowed the selective extraction of soluble phenols and eliminated interference by lipid-soluble pigments. Absorbance of the extracts between 260 to 560 nm was determined at 1 nm intervals and adjusted to unit frond area (Absnm cm–2).
Chlorophylls and carotenoids were estimated by the methods of Knudson et al. (1977) and Jaspers (1965). Absorbance measurements were taken at 450, 649, and 665 nm. Chlorophylls and carotenoids were expressed on a unit area basis (µg cm–2).
Growth
After pigment extraction the fronds were stained, mounted, and their areas determined. The plants were covered with a small volume (c. 0.5 ml) of 50% ethanol and two drops of 2% aqueous o-toluidine blue, allowed to stand overnight, rinsed three times with 10 ml aliquots of 50% ethanol, and transferred to dishes where the roots were removed and the individual fronds were teased apart. Individual fronds were arranged on a glass slide, blotted, and transferred to labelled 4x4 cm Whatman No. 3 filter paper squares with clear urethane packing tape. The resulting laminated mounts were placed beneath an incandescent lamp to warm them gently and to speed drying. After 4–6 h under the lamp the mounts were placed in a press until drying was complete. Digital images of the stained mounts were made on a flatbed scanner at 100 pixels cm–1 and frond areas determined using image analysis software (SigmaScan, Jandel Scientific Corp., San Rafael, CA).
Fresh and dry weights of the duckweed cultures were determined. The contents of culture flasks were sieved, rinsed, and blotted to remove as much water as practical. After recording the fresh weight the samples were allowed to dry at 60 °C until no further water loss was observed and the dry weight recorded.
UV treatments and radiation measurement
UV-B radiation was supplied by four fluorescent lamps (UV-313, Q-Panel Co., Cleveland, Ohio) suspended above the laboratory bench. Radiation was quantified with a UV-X broadband digital UV radiometer (UVP Inc., San Gabriel, CA) calibrated to this study’s system’s spectral distribution with an OL754 spectroradiometer calibrated with an OL752–10 NIST traceable 200 W tungsten-halogen standard lamp (Optronic Laboratories, Inc., Orlando, FL). At harvest, duckweed colonies were transferred to trays filled with distilled water to a depth of about 1.5 cm and covered with solarized cellulose diacetate film to cut off wavelengths <290 nm and to increase the humidity next to the plants. The covered trays were then placed under the fluorescent sunlamps which delivered a biologically effective ultraviolet flux of 2 W m–2 using the generalized plant action response spectrum normalized to 300 nm (Caldwell, 1971) for 15, 30, 45, 60, or 75 min.
Because the UV-313/cellulose diacetate (313CA) lamp/filter combination used for the UV-B treatments delivered some UV-A (315–400 nm) radiation (Fig. 1), a separate experiment was done to assess the effects of the UV-A radiation. These plants were irradiated for 1 h with the UV-313 lamps, but radiation was filtered through polyester film spectrally equivalent to Mylar type D plastic to remove wavelengths shorter than 313 nm (313Poly). To compare the relative action of UV-A radiation to the UV-B treatment, a UV340/Polyester (340Poly) lamp/filter combination was used, and the lamp height was adjusted so that the radiation delivered was comparable to that of the 313CA. With this method, similar peak irradiances and total power were delivered (Fig. 1). The total UV energy delivered by the 313CA lamp filter combination was 17.97 W m–2. The total UV energy delivered by the 340Poly lamp combination was 18.00 W m–2.
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Chlorophyll fluorescence
Immediately after irradiation, duckweed colonies were placed in chambers configured to permit gas exchange while maintaining an atmosphere saturated with water vapour. After a 2 h dark-adaptation period, the effect of UV-B radiation on the photochemical efficiency of PSII was determined as the relative variable chlorophyll fluorescence (Fv/Fm) using a CF-1000 steady-state chlorophyll fluorescence kinetics measurement system (Morgan Scientific Inc., Andover, MA) delivering 500 µmol m–2 s–1 PAR over 10 s.
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Growth
Frond areas were significantly reduced by AIP treatments at both levels compared with the controls (Multiway Bonferroni t-test, P <0.05). Although fresh weights were reduced by AIP, dry weights were not affected (Fig. 2).
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Scanning electron microscopy
SEM microscopy (Fig. 3) revealed that aerenchyma development was altered by AIP. The observed reduction in frond area is probably due to poorly differentiated aerenchyma and thicker fronds caused by AIP treatment.
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Optical microscopy
Ammonia (Fig. 4a–c) induced a yellow-green fluorescence characteristic of sinapoyl or feruloyl esters and intensified blue fluorescence attributable to coumaroyl esters (Hartley and Harris, 1980). Faint blue fluorescence associated with the cuticle was not eliminated by AIP. Differences in localization of these compounds were found between control plants and those grown with 0.5 µM AIP. In control plants, this fluorescence was associated with both the epidermis and upper mesophyll. In plants grown on 0.5 µM AIP, the fluorescence was mostly restricted to the upper mesophyll cells immediately adjacent to the epidermis. A similar pattern of phenolic expression and inhibition was revealed by UV epifluorescent visualization with both NP (Fig. 4d–f) and Zr (Fig. 4g–i) histochemicals.
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Sections treated with natural products reagent and viewed under UV (Fig. 4d–f) exhibited yellow fluorescence characteristic of NP-flavonoid complexation (Hutzler et al., 1998). Overall, AIP caused reductions in fluorescence in a dose-dependent manner. Although this fluorescence was visible throughout the mesophyll in control plants, it was most intense towards the upper epidermis (Fig. 4d). Plants grown on 10 µM AIP showed near-total elimination of fluorescence associated with secondary phenols with the exception of a very faint blue fluorescence primarily associated with the cuticle (Fig. 4f) and some localized yellow fluorescing regions scattered throughout the upper epidermis. In some samples, a very faint blue fluorescence was seen in the lower epidermis with NP. It should be noted that NP epifluorescence microscopy was the most sensitive technique applied to detect phenolics, and many fronds examined only exhibited chlorophyll fluorescence with no evidence of flavonoids. Since residual secondary phenols could conceivably be eluted from the tissues by the ethanolic NP reagent it was thought best to approach such results with caution and check results from the NP reagent with those from the aqueous Zr reagent.
Sections treated with 1% zirconium chloride (Fig. 4g–i) appeared generally similar to those treated with the NP reagent, but were crisper and lacked the ‘smearing’ caused by NP in the ethanolic carrier. NP treatment typically caused chloroplasts to appear to fluoresce an intense yellow. This may have resulted from increasing the permeability or rupturing of the tonoplast allowing vacuolar flavonoids to contaminate plastids. Some light scattering may have occurred, confusing the results somewhat; the NP reagent caused very intense fluorescence (Fig. 4d). By contrast, Zr fluorescence was restricted to the periphery of cells, either the cell walls or very near the tonoplast. In Zr-treated sections of control plants (Fig. 4g), vasculature fluorescence was more intense relative to that of the mesophyll. By comparison, NP treatments only elicited a light blue fluorescence typical of phenylpropanoids.
UV-visible spectroscopy
Absorbance spectra of aqueous 50% methanolic extracts in the UV-B are shown in Fig. 5. Treatment with 0.5 and 10.0 µM AIP resulted in reductions of 58% and 70%, respectively. Although the shapes of the absorbance spectra of control plants and those grown on 0.5 µM AIP are similar, absorbance was not equally affected at each wavelength. Anthocyanins, which exhibited a peak absorbance at about 525 nm were nearly totally eliminated by 0.5 µM AIP. (In control plants, anthocyanin pigmentation appeared only on the lower surface of the fronds so their potential contribution to UV screening was negligible.) Decreased absorbance was highly significant (P <0.001, Student’s t-test) at each wavelength within the UV-B waveband and throughout much of the UV-A waveband. Absorbance maxima characteristic of photosynthetic pigments were not detected in these extracts.
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Chlorophylls and carotenoids
Significant changes in total chlorophylls and carotenoids expressed on a frond area basis were observed in response to AIP, but no clear pattern emerged (Fig. 6). This might be linked to the increase in thickness and decrease in frond areas. The ratios of chlorophylls a/b and carotenoids to chlorophylls exhibited only slight increases.
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Fluorescence kinetics
AIP had no effect on PSII quantum efficiency (as Fv/Fm) of dark-adapted duckweed receiving no UV-B treatments. UV-B treatments had no significant effect on plants grown in the absence of AIP. However, AIP-treated plants were extremely sensitive to UV-B irradiation (Fig. 7). Plants grown in the presence of 0.5 µM AIP were relatively insensitive to low levels of UV-A, although high levels delivered by the UV-A lamps (340Poly) did depress Fv/Fm (Fig. 8). Plants grown at the 10.0 µM level were much more sensitive to UV-A radiation and showed significant effect even with the 313Poly lamp/filter combination. In every case, however, the UV-B treatments (313CA) elicited the greatest response.
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Discussion |
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AIP, is a highly specific chemical inhibitor of the enzyme PAL, and was used in an attempt to produce S. intermedia plants devoid of secondary phenolics. As the largest of the duckweeds, its relatively large fronds facilitate determination of physiological and biophysical parameters at the whole organ level. Since its natural growth habit is to float prostrate on the surface of the water, it is believed that blocking lignification of tracheary elements did not lead to adverse effects on xylem transport or compromise the plant’s ability to support itself, as might be expected with a terrestrial plant grown with AIP.
Spectroscopic analysis of methanolic frond extracts was used to assay AIP-induced inhibition of soluble, presumably vacuolar, phenols. Epifluorescence microscopy was used as an in situ assay of phenolic inhibition and to examine localization of secondary phenols. Since it was thought that membrane permeability might be enhanced by the ethanolic NP reagent and confound localization of soluble phenolics, Zr was also used to examine phenolic UV fluorescence. The overall pattern of fluorescence induced by Zr, NP, and NH3, were generally similar. Fuming plant material with NH3 has been used to elicit a generalized enhancement of phenolic fluorescence (Hartley and Harris, 1980). Bright yellow or orange fluorescence in response to NP is considered diagnostic for flavonoids (Hutzler et al., 1998). Overall, the fluorescence of Zr-treated sections was not as intense as NP; and Zr consistently failed to detect the upper epidermal phenols occasionally observed with NP in plants grown with 10 µM AIP.
The binding of metals by flavonoids has been known for well over a century (Welcher, 1948, and references therein). A wide range of flavonols have demonstrated affinities for heavy metals (Katyal and Prakash, 1977). Zirconium and other metal salts, were once widely used for the characterization and UV fluorescent visualization of flavonols on planar chromatograms, but have apparently fallen into disuse because of the lower detection limits achieved with NP (Gage et al., 1951; Seikel, 1962; Geiger, 1985). It has been demonstrated that zirconyl chloride is also potentially useful as a histochemical stain for UV epifluorescent microscopic localization of secondary phenols in plant tissues. Conversely, this method also allowed in situ visualization of regions capable of binding the metal by phenolic chelation.
Taken together, the results of spectroscopic analysis of frond extracts and UV-epifluorescence microscopy unambiguously demonstrated the inhibition of secondary phenolic synthesis by AIP. Secondary phenols have been implicated in a wide range of plant environmental and developmental interactions not limited to UV stress responses. For example, phenols have been implicated as required oxidase co-factors in auxin catabolism (Stafford, 1991), regulators of auxin transport (Brown et al., 2001), in vacuolar metal sequestering through chelation (Brouillard, 1988; Hale et al., 2002), and as a structural component of the primary cell wall matrix (Hatfield et al., 1999). The ability to grow plants devoid of secondary phenolics for several vegetative generations could yield important insights with regard to these, and other processes. Thus a secondary goal of this work was to develop, and at least partially characterize, one system with which to investigate these varied hypotheses further.
Wall-bound hydroxycinnamic acids are structural components in many plants and have been implicated in reducing wall extensibility (Liu et al., 1995). Thus, it was thought that PAL inhibition by AIP would result in normal-sized or slightly larger plants. Specifically, it was expected that either there would be no change in the frond area or that a slight isotropic frond expansion of the AIP-treated plants, resulting from a generalized, relatively consistent increase in cell expansion throughout the fronds, would be seen, especially since increased cotyledon area was observed when red cabbage was germinated in the presence of AIP (Gitz III et al., 1998). However, significant fresh weight and area reductions were observed with both AIP levels investigated, inconsistent with this hypothesis. Further, reduction in frond size was anisotropic and associated with obvious increases in frond thickness. Aerenchyma chambers were consistently smaller and irregularly distributed lending a ‘compressed’ appearance to the frond sections (Fig. 3). The increased frond thickness along with the compressed aerenchyma and reduced area may reflect a relative decrease in the lateral epidermal expansion with respect to that of the mesophyll cells; or an inability of the mesophyll to overcome the wall-bound phenolic-mediated epidermal yield threshold, resulting in bulging of the fronds (rather than lateral expansion). This is consistent with the results of histochemical analysis of 10 µM AIP-grown plants which revealed that some fluorescence, typical of wall-bound phenols, was present in the epidermis, but nearly completely eliminated in the mesophyll. In fronds examined by SEM, mesophyll cell size apparently increased, though this was not quantified. The observed increase in cell size and decrease in fresh weight of cultures is consistent with the general observation that inhibition of cell wall synthesis is typically associated with tissue swelling and reduced cell division (Abeles et al., 1973).
Ethylene is known to elicit tissue swelling, or gibbosity, in Lemna gibba and Lemna minor (Elzenga et al., 1980; Cleland and Tanaka, 1982). However, there appear to be no reports of ethylene induction from PAL inhibition. In fact, two other PAL inhibitors, -aminooxyacetic acid (AOA) and L--aminooxy-ß-phenylpropionic acid (AOPP), inhibited ethylene synthesis (Amrhein and Wenker, 1979). At any rate, ethylene-induced gibbosity typically results in larger, well-formed lacunae; while lowered ethylene levels result in a flatter frond with well-formed lacunae (Cleland and Tanaka, 1982). Clearly, neither of these ethylene responses were evident in the present case, although ethylene responses cannot be conclusively ruled out.
The observed morphological changes might also be explained by altered auxin content. Oxidase-mediated auxin catabolism is enhanced in vitro by certain phenylpropanoids and flavonoids which act as required co-factors and it has been suggested that flavonoids may have originally evolved as growth regulators (Stafford, 1991). Recent work has provided direct evidence of such a regulatory role for flavonoids in Arabidopsis (Brown et al., 2001). A link between oxidase expression, auxin degradation, and UV-B tolerance has been established in duckweed (Jansen et al., 2001). It has been suggested that UV-B-elicted photomorphogenic responses result, in part, from oxidative auxin degradation and altered flavonol expression (Jansen, 2002). Likewise, in the present work, differences in phenolic content might have led to differences in rates of auxin catabolism within the fronds. Since AIP is a competitive inhibitor of PAL, although an extremely effective one, PAL activity cannot be completely eliminated. While traces of phenylpropanoids from residual PAL activity were found in the epidermis, mesophyll phenols were apparently eliminated (Figs 4f, 4i, 5c). Early reports of growth responses of duckweed to exogenous IAA include frond epinasty and reduced root growth (Hillman, 1961). Reduced root growth was observed in response to 10 µM AIP during the course of this experimentation (not shown). It is suggested that the morphological changes observed in the mesophyll of duckweed grown with 10 µM AIP resulted (Fig. 3b) from an indirect effect of AIP leading to increased auxin levels.
It is not known whether AIP affected the expression of peroxidases having IAA oxidase activity, since they were not assayed during these experiments. However, in the few plants which have been investigated, AIP has not been shown to affect pool sizes of soluble amino acids other than phenylalanine, which increased (Zon and Amrhein, 1992). Indirect evidence from the current investigation support these observations: dry weights were unchanged by AIP treatments (Fig. 2), so at least basal metabolism was unaffected by the inhibitor. This is in contrast to the report in which AIP treatment actually increased the culture dry mass of the duckweed Spirodela punctata while simultaneously decreasing multiplication rate over the course of 8 d (Janas et al., 1998).
Decreases in Fv/Fm values are commonly interpreted as early signs of PSII stress. The UV-B sensitivity of the D1/D2 heterodimer protein core of the photosystem II complex has been known for some time. The D1 protein has been shown to be especially sensitive to UV-B although destruction of D2 and light-harvesting antenna proteins also occur, especially with background white light. It has been demonstrated that UV-A radiation is relatively ineffective in eliciting PSII degradation in duckweed (Jansen et al., 1996a, b). In the present work it was found that, with greatly reduced phenolic accumulation, UV-A radiation was effective in reducing Fv/Fm (Fig. 8) although it was not nearly as damaging as UV-B. The absorptivity of the extractable phenols does extend to the UV-A (Fig. 6) and UV sensitivity of isolated spinach thylakoids and the green alga Dunaliella salina extends into the UV-A waveband (Bornman et al., 1984; Ghetti et al., 1999). Arabidopsis transparent testa mutants unable to synthesize flavonols were found to be sensitive to UV-A radiation (Fiscus and Booker, 2002). In normally developing duckweed it is difficult to demonstrate such sensitivity (Jansen et al., 1996b). This underscores the view that differences in anatomy and phenolic expression between species affects radiation distribution within tissues and UV tolerance.
Although there was a significant response to AIP in total chlorophylls and carotenoids, no clear pattern emerged and it is thought that these results may be linked to the morphological changes noted above. Increases in leaf thickness and carotenoids are generally thought to lower the sensitivity of PSII to UV-B (Jansen et al., 1998, 1996b). Even with the consistent, although slight, increases in the carotenoid to chlorophyll ratios and the increased frond thickness resulting from growth on 10 µM AIP, the sensitivity to UV-B increased with reduced phenolic content. Thus, these effects are separable from the phenolic-dependent responsiveness of Fv/Fm to UV-B observed in these experiments. All of the results in this work are consistent with, and provide further direct evidence of the working hypothesis that phenols function to screen UV radiation before reaching photosynthetic tissues in these plants.
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Conclusions |
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This work provides further direct evidence of the role of secondary phenols in conferring UV tolerance in a duckweed without resorting to the generation and characterization of mutants. However, normally developing plants with a full complement of UV-absorbing compounds were resistant to UV radiation.
Additional evidence was found consistent with the concept of regulation of auxin catabolism or transport by flavonoids and perhaps other phenols in duckweed.
The use of zirconium for visualization of phenols takes advantage of UV-induced fluorescence of metal–flavonol complexes through well-understood mechanisms, and was useful as a novel histochemical technique to reveal phenols within these tissues.
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References |
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